The primary goal of this project is to use cryo-EM single particle analysis to obtain higher resolution structures (6-10E) of the anthrax toxin protective antigen (PA) pore complex inserted into lipid nanodiscs. Accumulating PA pore nanodisc image data sets using cryo EM techniques will allow us to obtain higher resolution structural detail of the PA-pore -nanodisc complex (6-10 E). Normal mode flexible fitting procedures have allowed us to determine the location of the flexible loop containing the phe427 clamp. We will use this method to specifically define the important pH dependent dynamic conformational modes involved in protein translocation as we determine the higher resolution structures of the PA pore nanodisc structure. Specifically, we shall examine the changes that occur in the PA pore nanodisc complexes 1) as pH changes from 7.0 to 5.5, 2) We shall compare the wild type PA pore nanodisc structures with the PA F427A translocation mutant nanodisc as a function of pH. 3) We shall determine the complexes of the PA pore nanodisc bound to the full length lethal factor and the N terminal domain. These comparative structural approaches will enable us to define structural elements involved in translocation control for projects 1 and 2. For project 3 our success at constructing PA pore nanodisc complexes gives us the unique ability to investigate a possible structural change in PA pore and possibly in PA pore-lethal factor complexes when the pH is decreased from 7 to 5.5, mimicking changes in binding interactions and transitions that occur during endosome acidification. Research and Biotechnology implications: The nanodisc-anthrax toxin pore complex may serve as a specific antigen delivery system for the active form of this toxin component. Folding membrane proteins under immobilizing conditions prevents aggregation but allows us to easily insert these properly folded proteins into lipid nanodiscs or other lipid structures thus aiding membrane protein structure determination. Our methods may serve as a basis model method for constructing and studying other bacterial toxin macromolecular complexes (e.g. Clostridia Toxin). Nanodisc complexes will enable us to probe and control kinetic and dynamics of binding interactions for large populations or single molecules. These particular complexes could serve as platforms for the development of high throughput screening platforms to identify anti-anthrax prophylactics. PUBLIC HEALTH RELEVANCE: Inhalation of the Bacillus anthracis spores, a potential bioweapon and subsequent production of exotoxin causes severe toxemia, bacteremia and death. We are implementing novel methods to isolate and view the structure of the anthrax toxin translocation pore complex (Protective Antigen) to apply this new structural knowledge toward developing new therapeutic anti-anthrax treatments and novel vaccines.